Avidin is a basic glycoprotein derived from albumen and strongly binds to biotin (vitamin H). On the other hand, streptavidin is an avidin-like protein derived from Streptomyces avidinii and has an approximately neutral isoelectric point and does not have a sugar chain. Both proteins form tetramers, and one subunit binds to one biotin molecule. The molecular weights are about 60 kDa. The affinity of avidin to biotin or of streptavidin to biotin is very high (Kd=10−15 to 10−14 M) and is the highest as interaction between two biomolecules. Accordingly, avidin/streptavidin-biotin interaction has been widely used in the fields of biochemistry, molecular biology, and medicine. Avidin has an isoelectric point higher than 10, and this high basicity or the presence of a sugar chain problematically causes non-specific binding to biomolecules, such as DNA and protein, in some cases.
Biotin has a small molecular weight of 244 and is stable for a change in pH and heat and, therefore, is commonly used as a label of substances. In a method of biotinylation, chemically modified biotin is bound to a functional group of protein, such as an amino group, a carboxyl group, or an aldehyde group. Biotinylating reagents are commercially available and can be used to biotinylate protein, nucleic acid, and other substances. In a method of biotinylating protein, a fusion protein of a target protein and a sequence that will be biotinylated by biotin ligase in vivo is expressed as a recombinant protein, and the resulting fusion protein is biotinylated by the biotin ligase in a host cell. For example, BIOEASE TAG™ (biotinylated sequence) is a biotinylated sequence supplied by Life Technologies Corporation and is on the market as a system for expressing a biotinylated protein in vivo, in E. coli, drosophila, or mammal cells.
The binding between avidin or streptavidin and biotin is significantly strong and is thus irreversible, and the both are hardly dissociated from each other after the binding is formed once. Because of this strong binding, known avidin and streptavidin cannot be directly applied to technical fields that require reversible binding, such as affinity chromatography, for purifying biotinylated biomolecules.
Countermeasures which have been reported against this problem are avidin and streptavidin showing reduced biotin-binding affinity. For example, nitrated avidin and nitrated streptavidin in which the tyrosine residue contributing to binding to biotin is nitrated have been developed. They strongly bind to biotin under acidic to neutral conditions (pH 4 to 7.5) and are dissociated from biotin under alkaline conditions (pH 10). Nitrated avidin agarose is commercially available as CAPTAVIDIN-AGAROSE™ (nitrated avidin agarose). However, nitration is a troublesome task, and its efficiency is not constant. In addition, an extreme change in pH may adversely affect biotinylated protein and so on.
At the same time, it has been reported to reduce the affinity to biotin by introducing site-specific amino acid mutation to avidin or streptavidin through genetic engineering. Two methods are known for reducing affinity to biotin: a method of introducing a modification into an amino acid that directly interacts with biotin among amino acids forming a biotin-binding pocket; and a method of introducing a modification into an amino acid that is involved in the interaction between subunits of the protein.
In the case of avidin, recombinant proteins having reduced affinity to biotin have been reported in which a modification is introduced to the amino acid that forms a hydrogen bond with biotin (Marttila, et al., (2003), Biochem. J., 369: 249-254; Laitinen, et al., (2003), J. Biol. Chem., 278: 4010-4014; Laitinen, et al., (2001), J. Biol. Chem., 276: 8219-8224) or a modification is introduced to the amino acid that forms a hydrophobic bond with biotin (Laitinen, et al., (1999) FEBS Lett., 461: 52-58; Laitinen, et al., (2003), J. Biol. Chem., 278: 4010-4014).
Similarly, in the case of streptavidin, examples are known in which a modification is introduced to the amino acid that forms a hydrogen bond with biotin (Qureshi, et al., (2001), J. Biol. Chem., 276: 46422-46428; Gabriel, et al., (1998), Proc. Natl. Acad. Sci., 95: 13525-13530; Qureshi and Wong, (2002), Protein Expr. Purif., 25: 409-415; Wu and Wong, (2006), Protein Expr. Purif., 46: 268-273; Wu and Wong, (2005), J. Biol. Chem., 280: 23225-23231) or a modification is introduced to the amino acid that forms a hydrophobic bond with biotin (Chilkoti, et al., (1995), Proc. Natl. Acad. Sci., 92: 1754-1758; Laitinen, et al., (1999), FEBS Lett., 461: 52-58; Sano, et al., (1995), Proc. Natl. Acad. Sci., 92: 3180-3184; Sano, et al., (1997), Proc. Natl. Acad. Sci., 94: 6153-6158).
Furthermore, in the cases of avidin and streptavidin, it has been reported to produce monomers of these proteins that are modified to reduce the affinity to biotin by introducing the modification to the amino acids involving in the interaction between subunits of these proteins (Laitinen, et al., (2001), J. Biol. Chem., 276: 8219-8224; Wu and Wong, (2005), J. Biol. Chem., 280: 23225-23231). Avidin and streptavidin each form a tetramer, and each subunit has one biotin-binding site. In order to form a complete biotin-binding pocket, the amino acid residue present in the adjacent subunit (for example, in the case of tamavidin 2, the 108th tryptophan (W108)) is important. Accordingly, it is believed that the binding between subunits also highly affect the affinity to biotin.
According to Wu, et al, (J. Biol. Chem., (2005), 280: 23225-23231), in the case of subunits of streptavidin designated as A, B, C, and D, the 55th valine of subunit A is present near the 59th arginine of subunit B. The 76th threonine of subunit A is present very close to the 76th threonine and the 59th alanine of subunit B. The 109th leucine of subunit B interacts with the 125th valine of subunit A. The 125th valine of subunit A widely interacts with the 109th leucine, the 120th tryptophan, the 123rd threonine, and 125th valine of subunit D, the 109th leucine of subunit B, and 107th glutamine of subunit C. Accordingly, charge repulsion or steric hindrance between subunits are expected to be generated through replacing these amino acids with highly polar amino acids such as arginine, lysine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and threonine. It is conceivable that arginine having the lowest hydrophaty index among these polar amino acids is particularly effective.
In order to apply the biotin-binding protein such as avidin and streptavidin to the technical field that requires reversible binding, such as affinity chromatography, a possible goal is to increase the dissociation constant (KD) to about 10−7 (M). Though depending on circumstance, in general, a dissociation constant less than this level leads to high biotin-binding ability that precludes efficient dissociation of a desired biotinylated substance, while a dissociation constant higher than this level leads to low biotin-binding ability that precludes sufficient binding of a desired biotinylated substance (Wu and Wong, (2006), Protein Expr. Purif., 46: 268-27).
In light of these points, among the streptavidin mutants, every single-amino acid mutant at the hydrogen bond site has a low dissociation constant of about 10−11 (M) and significantly high biotin-binding ability. However, many of these mutants have an effect on the interaction between subunits by amino acid modification to often give monomers. In the case of the monomers, the dissociation constant is about 10−9 (M). Furthermore, among the streptavidin mutants, if two or more hydrogen bond sites are further modified, the tetramers are mostly dissociated into monomers, some of these monomers have a biotin-binding ability (dissociation constant) of about 10−8 to 10−6 (M) (Qureshi, et al., (2001), J. Biol. Chem., 276: 46422-46428).
Mutants having a dissociation constant of 10−8 to 10−7 (M) have biotin-binding ability suitable for application to, for example, affinity chromatography (Qureshi and Wong, (2002), Protein Expr. Purif., 25: 409-415; Wu and Wong, (2006), Protein Expr. Purif., 46: 268-273). However, these monomers are known to be easily decomposed by proteases (Laitinen, et al., (2001), J. Biol. Chem., 276: 8219-8224; Wu and Wong, (2005), J. Biol. Chem., 280: 23225-23231). Affinity chromatography often uses a crude cell extract containing various substances. Many of such crude cell extracts contain proteases to cause a problem when the monomers are used in such application.
In addition, in the monomers, the hydrophobic region that is hidden by the binding between subunits is exposed, which probably reduces the overall solubility of the protein and may cause reaggregation. In monomers designed using avidin as a model (for example, SOFTLINK™ Soft Release Avidin Resin (resin to which monomeric avidin as a model is immobilized), available from Promega Corp.), the monomers associate with one another to form a tetramer when they are immobilized to a carrier. As a result, the affinity with biotin is increased. Accordingly, it is necessary to fill the region of the tetramer that strongly binds to biotin with biotin before addition of as biotin-labeled substance. This treatment is a troublesome task and may highly affect the yield of the biotin-labeled substance depending on the degree of the pretreatment.
In a very small number of streptavidin mutants, the tetramer form is maintained even if amino acids at two positions of a hydrogen bond site are modified. However, in such a tetramer, the interaction between subunits is weakened due to the modification, and a phenomenon in which many of the monomers constituting the tetramer are dissolved is observed after a biotinylated substance bound to the tetramer immobilized to a carrier is eluted by adding an excess amount of biotin thereto.
Furthermore, many amino acid-modified proteins of avidin and streptavidin cannot be solubly expressed in E. coli and have to be expressed in insect cells or Bacillus subtilis cells (Laitinen, et al., (1999), FEBS Lett., 461: 52-58; Qureshi and Wong, (2002), Protein Expr. Purif., 25: 409-415), which raises labor and cost issues. Only some monomeric streptavidins can be solubly expressed in E. coli (Wu and Wong, (2006), Protein Expr. Purif., 46: 268-273).
As described above, it has not yet been known as biotin-binding protein that has biotin-binding ability allowing the protein to sufficiently bind to and to be dissociated from a desired biotinylated substance, that can be solubly expressed in E. coli, and that has protease resistance.
The present inventors have discovered tamavidin 1 and tamavidin 2, which are novel avidin-like biotin-binding proteins, in an edible mushroom (Pueurotus conucopiae) (WO02/072817). Tamavidin 1 and tamavidin 2 can be expressed in E. coli. In particular, tamavidin 2 can be easily prepared by purification using an iminobiotin column (WO02/072817). Tamavidin 1 and tamavidin 2 extremely strongly bind to biotin. In particular, tamavidin 2 shows a biotin-binding activity almost equal to that of avidin or streptavidin. Furthermore, tamavidin 2 is a biotin-binding protein excellent in that the heat resistance is higher than that of avidin or streptavidin and that the non-specific binding is less than that of avidin.